Ibuprofen is a common over-the-counter drug taken for pain relief. However, the pig may provide a useful model where extensive amide hydrolysis is seen for drugs or environmental chemicals in humans, but not in e.g., the rat and dog which are the pre-clinical species normally employed for safety assessment. Given its low sulfation, combined with significant capacity for N-deacetylation the pig may represent a poor translational model for toxicology studies for compounds undergoing significant metabolism by sulfation, or which contain amide bonds which when hydrolysed to unmask an aniline lead to toxicity. The phenolic sulfate of APAP, and the glutathione-derived metabolites of the drug were only found as minor components (with the cysteinyl conjugate detected but not the mercapturate). The PAP produced by N-deacetylation was the likely cause of the methaemoglobinemia and kidney toxicity observed in this, and previous, studies in the pig. The major circulating and urinary metabolite of APAP was the phenolic glucuronide (APAP-G), followed by p-aminophenol glucuronide (PAP-G) formed from N-deacetylated APAP. A specific liquid chromatography-tandem mass spectrometry assay was used to quantify APAP and metabolites. Increased concentrations of citrate combined with pre-ALF increases in circulating lactate, pyruvate and alanine in plasma suggest mitochondrial dysfunction and a switch in hepatic energy metabolism to glycolysis in response to APAP treatment. Studies were undertaken to determine both the metabolic fate of APAP and its effects on the endogenous metabolic phenotype of ALF in using 1H NMR spectroscopy. Anaesthetized pigs were initially dosed at 250 mg/kg via an oroduodenal tube with APAP serum concentrations maintained above 300 mg/L using maintenance doses of 0.5-4g/h until ALF. The metabolic fate, toxicity and effects on endogenous metabolism of paracetamol (acetaminophen, APAP) in 22 female Landrace cross large white pigs were evaluated in a model of acute liver failure (ALF). In conclusion, we show that APAP toxicity in HepG2 cells is largely independent of oxidative stress, and is linked instead to a decoupling of glycolysis from the TCA cycle, lactic acidosis, reduced NADPH production, and subsequent suppression of the anabolic pathways required for rapid growth. Co-treatment with a specific inhibitor of the lactate/H⁺ transporter MCT1, AZD3965, led to increased apoptosis in APAP-treated cells, suggesting that lactate accumulation could be a cause of cell death in this model. These events were not associated with lower reduced glutathione levels and no glutathione conjugates were seen in cell extracts. This was accompanied by a depletion of cellular NADPH and a reduction in the de novo synthesis of fatty acids and the amino acids serine and glycine. Metabolically, we observed an up-regulation of glycolysis and lactate production with a concomitant reduction in carbon from glucose entering the pentose-phosphate pathway and the TCA cycle.

We found that APAP exposure leads to limited cellular death and substantial growth inhibition. In this study, we employed metabolic profiling and isotope labelling approaches to investigate the metabolic impact of acute exposure to cytotoxic doses of APAP on the widely used HepG2 cell model. Hepatoma cell lines typically lack the CYP450 activity to generate the reactive metabolite of APAP observed in vivo, but are still subject to APAP cytotoxicity. Acetaminophen (APAP) is one of the most commonly used analgesics worldwide, and overdoses are associated with lactic acidosis, hepatocyte toxicity, and acute liver failure due to oxidative stress and mitochondrial dysfunction.

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